Project Summary/Abstract Insult to the auditory system often results in irreparable hearing loss due to the inability of mature mammalian hair cells to regenerate (Burns, J.C. and Corwin, J.T. 2013; Groves, A.K. 2010). However, evidence suggests that minor damage to mechanotransductive hair bundles can be repaired in some cases. For example, gaps in phalloidin labeling of the F-actin in stereocilia are found after mechanical damage and appear to indicate filament breaks. Factors important for the nucleation, elongation, and crosslinking of F-actin assembly, including ?-actin, cofilin, and espin, are enriched in these sites (Belyantseva, I.A. et al. 2009). My proposal aims to investigate the mechanisms mediating this F-actin repair process. In Specific Aim 1, I will test the hypothesis that damage of stereociliar F-actin leads to the incorporation of newly synthesized F-actin in stereocilia. In order to accomplish this, I will develop mouse models for the visualization of the response of newly synthesized actin to mechanical damage, using the FLEx-Cre switch system. Repair of the F-actin core is expected to depend on proteins that mediate the nucleation, polymerization, stabilization and/or crosslinking of actin filaments. My preliminary studies suggest that XIRP2 (Xin Actin Binding Repeat Containing 2) may be involved in the stereocilia F-actin repair process, as well. We previously described XIRP2 as a novel hair cell protein enriched in the hair bundle, where it colocalizes with F-actin. Knockout of Xirp2 causes progressive hearing loss in mice (Francis, S.P. et al. 2015); moreover, heterozygous mutations in XIRP2 were identified in humans with age related hearing loss. While less severe than the hearing loss in the total Xirp2 knockout mice, heterozygous Xirp2 mice also develop progressive hearing loss. Additionally, immunostaining shows the XIRP2 is enriched in gaps in phalloidin staining, similar to those described above. In Specific Aim 2, I will test the hypothesis that XIRP2 is recruited to these gaps in as a direct response of mechanical damage. At these sites, XIRP2 may facilitate the maintenance or repair of the F-actin stereocilia core and I expect that the dysfunctional repair process in Xirp2-null mice leads to the observed progressive decline in hearing function. In order to visualize the response of XIRP2 to hair bundle damage, I will knock in a fluorescent tag at the endogenous Xirp2 locus to enable live imaging. I will determine whether XIRP2 is recruited to damaged sites in F-actin stereocilia cores using both a mechanical and genetic model of damage.